Method for measuring hematocrit value of blood sample, method for measuring concentration of analyte in blood sample, sensor chip and sensor unit

ABSTRACT

Voltage is applied across a counter electrode and a working electrode, with which a blood sample is in contact, in such a state that an oxidant in a redox substance is not substantially in contact with a working electrode but is in contact with a counter electrode and a reductant is not substantially in contact with the counter electrode but is in contact with the working electrode, whereby the reductant and the oxidant are respectively oxidized and reduced to measure current produced upon the oxidation and reduction. According to the above constitution, while lowering the voltage applied across the working electrode and the counter electrode, the Hct value of the blood sample can be measured stably with a satisfactory detection sensitivity. This measurement can be carried out with a sensor chip comprising a working electrode ( 11 ), a counter electrode ( 12 ), and a blood sample holding part ( 14 ) having branch parts ( 18   a,    18   b ). A first reagent ( 13 ) consisting essentially of an oxidant in a redox substance is disposed in contact with the surface of a counter electrode which faces the branch part ( 18   b ), and a second reagent ( 19 ) consisting essentially of a reductant is disposed in contact with the surface of a working electrode that faces the branch part ( 18   a ).

TECHNICAL FIELD

The present invention relates to a method for measuring a hematocrit(Hct) value of a blood sample, a method for measuring a concentration ofan analyte in a blood sample, and a sensor chip and a sensor unit suitedfor such measurements.

BACKGROUND ART

Sensor chips have been used for the measurement of an analyteconcentration in a blood sample, for example, such as a blood glucoseconcentration (blood sugar level).

The sensor chip measures the amount of current flowing in the bloodsample after an enzymatic cycling reaction involving an analyte, and aconcentration of the analyte is calculated based on the measured currentamount. The amount of current varies not only with the analyteconcentration but the Hct value of the blood sample. The Hct value ofthe blood sample varies according to the physical condition of theanimal from which the blood sample is drawn. In humans, the Hct value isnormally 39% to 50% for adult males, and 36% to 45% for adult females.It is therefore desirable that the Hct value of the blood sample also bemeasured by the sensor chip in order to specify accurately the analyteconcentration in the blood sample, and to find the attributes of theblood sample, for example, such as blood viscosity and anemia.

Sensor chips for measuring the Hct value of a blood sample are disclosedin JP8(1996)-500190T, JP15(2003)-501627T, a pamphlet of InternationalPublication 2005/054839, and a pamphlet of International Publication2005/054840. These known sensor chips include an electrode systemequipped with a working electrode and a counter electrode, and a channel(blood sample holder) for holding a blood sample between the workingelectrode and counter electrode.

In the sensor chip of the JP8(1996)-500190T, a reductant and an oxidantof an electron mediator are disposed on the blood sample holder to bedissolved by a blood sample. The reductant and the oxidant of theelectron mediator mix with the blood sample introduced into the bloodsample holder, and adhere to the working and counter electrodes in themixture with the blood sample. In the sensor chip described inJP15(2003)-501627T, the oxidant of the electron mediator is disposed onthe working and counter electrodes. In these sensor chips, the Hct valueof the blood sample is specified by measuring the amount of current thatflows in the blood sample as a result of a redox reaction of theelectron mediator adhering to the electrodes.

In the sensor chips described in the pamphlet of InternationalPublication 2005/054839 and the pamphlet of International Publication2005/054840, the electron mediator is disposed only on the counterelectrode of the electrode system including the working and counterelectrodes for measuring a Hct value. In these sensor chips, a pureblood sample not containing the electron mediator contacts the workingelectrode following introduction of the blood sample into the bloodsample holder. In the sensor chips, movement of electrons occurs at theinterface of the blood sample and the working electrode as a result of aredox reaction of the blood components in the blood sample, for example,such as ascorbic acid, uric acid, and water. The electron mediatordisposed on the counter electrode is involved in the movement ofelectrons at the interface of the blood sample and the counterelectrode.

DISCLOSURE OF THE INVENTION

In the sensor chips described in JP8(1996)-500190T andJP15(2003)-501627T, the amount of current (redox current) that flows inthe blood sample in the measurement of Hct value varies only slightlywith respect to a rate of change of the Hct value of the blood sample.Accordingly, the detection sensitivity is not sufficient in these sensorchips. For example, in some cases, a change in amplitude of the redoxcurrent is only about 8% when a change in Hct value of the blood sampleis 20%. In the sensor chips described in the pamphlet of InternationalPublication 2005/054839 and the pamphlet of International Publication2005/054840, the amplitude of the redox current fluctuates over a widerange when the voltage (Hct value measuring voltage) applied across theworking electrode and the counter electrode in the measurement of Hctvalue is decreased. A stable Hct measurement in the blood sample wouldnot be possible in this case.

An object of the present invention is to provide a method for measuringa Hct value of a blood sample, a method for measuring a concentration ofan analyte in a blood sample, and a sensor chip and a sensor unit suitedfor such measurements, that are capable of stably measuring the Hctvalue of the blood sample with sufficient detection sensitivity even ata low Hct value measuring voltage.

The inventors of the present invention designed such a layout pattern ofthe redox substance disposed on a sensor chip that, followingintroduction of a blood sample, a reductant of the redox substance is incontact with the working electrode and an oxidant of the redox substanceis contact with the counter electrode while the other form of the redoxsubstance is substantially not in contact with the counter electrode orthe working electrode. The inventors found that, with such a layoutpattern, the amplitude fluctuations of the redox current can besuppressed and the rate of amplitude change of the redox current withrespect to a change in Hct value immediately after the voltageapplication can be increased, even when the voltage applied across theworking electrode, serving as the anode, and the counter electrode,serving as the cathode, is decreased.

The present invention provides a method for electrochemically measuringa Hct value of a blood sample. The method includes: applying a voltageacross a working and a counter electrode in contact with the bloodsample; detecting a resulting current flowing between the workingelectrode and the counter electrode; and calculating a Hct value of theblood sample based on the current. The voltage is applied across theworking electrode and the counter electrode while an oxidant of a redoxsubstance is in contact with the counter electrode and substantially notin contact with the working electrode, and a reductant of the redoxsubstance in contact with the working electrode and substantially not incontact with the counter electrode. The current is detected by measuringa current that results from the oxidation of the reductant and thereduction of the oxidant caused by the voltage application.

In another aspect, the present invention provides a method forelectrochemically measuring a concentration of an analyte in a bloodsample. The method includes: electrochemically detecting a current Areflecting a Hct value of the blood sample so as to obtain data Arepresenting the current A or an equivalent of the current A andcorresponding to the Hct value; electrochemically detecting a current Bthat results from the oxidation or reduction of the analyte in the bloodsample caused, in the presence of a redox substance, by a redox enzymethat uses the analyte as a substrate, so as to obtain data Brepresenting the current B or an equivalent of the current B; anddetermining a concentration of the analyte in the blood sample based ondata C obtained by correcting the data B using the data A. The current Ais detected by detecting a current that results from the oxidation of areductant and the reduction of an oxidant of the redox substance causedby application of a voltage across a working electrode and a counterelectrode. The voltage is applied while the oxidant of the redoxsubstance is in contact with the counter electrode and substantially notin contact with the working electrode, and the reductant of the redoxsubstance is in contact with the working electrode and substantially notin contact with the counter electrode.

In another aspect, the present invention provides a sensor chipincluding a Hct value analyzer that electrochemically detects a currentreflecting a Hct value of a blood sample. The Hct value analyzerincludes: a working electrode and a counter electrode; a blood sampleholder for holding the blood sample in contact with the workingelectrode and the counter electrode; and a blood sample inlet throughwhich the blood sample is introduced into the blood sample holder. Afirst reagent containing an oxidant of a redox substance andsubstantially not containing a reductant of the redox substance isdisposed to cover a surface of the counter electrode facing the bloodsample holder. A second reagent containing the reductant andsubstantially not containing the oxidant is disposed to cover a surfaceof the working electrode facing the blood sample holder. In anotheraspect, the present invention discloses a sensor chip including a Hctvalue analyzer that electrochemically detects a current reflecting a Hctvalue of a blood sample. The Hct value analyzer includes: a workingelectrode and a counter electrode; a blood sample holder for holding theblood sample in contact with the working electrode and the counterelectrode; and a blood sample inlet through which the blood sample isintroduced into the blood sample holder. The blood sample holderincludes an inlet portion in communication with the blood sample inlet;and a first and a second branch portion branching out of the inletportion. The first branch portion faces the counter electrode, and thesecond branch portion faces the working electrode. A first reagentcontaining an oxidant of a redox substance and substantially notcontaining a reductant of the redox substance is disposed on the firstbranch portion. A second reagent containing the reductant andsubstantially not containing the oxidant is disposed on the secondbranch portion. In another aspect, the present invention provides asensor chip including a Hct value analyzer that electrochemicallydetects a current reflecting a Hct value of a blood sample. The Hctvalue analyzer includes: a working electrode and a counter electrode; ablood sample holder for holding the blood sample in contact with theworking electrode and the counter electrode; and a blood sample inletthrough which the blood sample is introduced into the blood sampleholder. The blood sample holder includes a first reagent, disposed onthe blood sample holder, containing an oxidant of a redox substance andsubstantially not containing a reductant of the redox substance, and asecond reagent, disposed on the blood sample holder, containing thereductant and substantially not containing the oxidant. One of the firstreagent and the second reagent is disposed on the upstream side of theother reagent with respect to a flow of the blood sample introduced intothe blood sample holder through the blood sample inlet. (a) When theother reagent is the first reagent, the counter electrode is disposed onthe downstream side of the working electrode with respect to the flow ofthe blood sample, and the first reagent is disposed to cover a surfaceof the counter electrode facing the blood sample holder, the workingelectrode is disposed on the upstream side of the counter electrode withrespect to the flow of the blood sample, and the second reagent isdisposed separately from the working electrode on the upstream side ofthe working electrode with respect to the flow of the blood sample, ordisposed in contact with the working electrode. (b) When the otherreagent is the second reagent, the working electrode is disposed on thedownstream side of the counter electrode with respect to the flow of theblood sample, and the second reagent is disposed to cover a surface ofthe working electrode facing the blood sample holder, the counterelectrode is disposed on the upstream side of the working electrode withrespect to the flow of the blood sample, and the second reagent isseparately disposed from the counter electrode on the upstream side ofthe counter electrode with respect to the flow of the blood sample, ordisposed in contact with the counter electrode.

Further, in another aspect, the present invention provides a sensor unitincluding the sensor chip, and a sensor main body including a voltageapplying circuit for applying a predetermined voltage across the workingelectrode and the counter electrode. The sensor chip is detachable withrespect to the sensor main body, and the voltage applying circuit iscapable of applying a predetermined voltage across the working electrodeand the counter electrode with the sensor chip attached to the sensormain body. The predetermined voltage is 3.0 V or less, when the workingelectrode is anode and the counter electrode is cathode.

According to the present invention, the Hct value of the blood samplestably can be measured with sufficient detection sensitivity, even at alow Hct value measuring voltage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view showing an example of a sensorchip for measuring a Hct value of the present invention.

FIG. 2 is a plan view showing an example of a sensor chip for measuringa Hct value of the present invention.

FIG. 3 is an exploded perspective view showing another example of asensor chip for measuring a Hct value of the present invention.

FIG. 4 is a plan view showing another example of a sensor chip formeasuring a Hct value of the present invention.

FIG. 5 is an exploded perspective view showing another example of asensor chip for measuring a Hct value of the present invention.

FIG. 6 is a plan view showing another example of a sensor chip formeasuring a Hct value of the present invention.

FIG. 7 is a graph representing an example of measurement results of Hctvalue by a sensor chip of Example 1.

FIG. 8 is a graph representing another example of measurement results ofHct value by the sensor chip of Example 1.

FIG. 9 is a graph representing another example of measurement results ofHct value by the sensor chip of Example 1.

FIG. 10 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 1.

FIG. 11 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 1.

FIG. 12 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 1.

FIG. 13 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 1.

FIG. 14 is a graph representing an example of measurement results of Hctvalue by a sensor chip of Example 2.

FIG. 15 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 2.

FIG. 16 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 2.

FIG. 17 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 2.

FIG. 18 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 2.

FIG. 19 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Example 2.

FIG. 20 is a graph representing an example of measurement results of Hctvalue by a sensor chip of Comparative Example 1.

FIG. 21 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Comparative Example 1.

FIG. 22 is a graph representing another example of measurement resultsof Hct value by the sensor chip of Comparative Example 1.

FIG. 23 is a graph representing an example of measurement results of Hctvalue by a sensor chip of Comparative Example 2.

FIG. 24 is a graph representing an example of measurement results of Hctvalue by a sensor chip of Comparative Example 3.

FIG. 25 is a graph representing an example of measurement results of Hctvalue by a sensor chip of Comparative Example 4.

FIG. 26 is a perspective view showing an example of a sensor unit formeasuring a Hct value of the present invention.

FIG. 27 is a diagram showing an example of a circuit structure of asensor unit for measuring a Hct value of the present invention.

FIG. 28 is an exploded perspective view showing another example of asensor chip for measuring a Hct value of the present invention.

FIG. 29 is a plan view showing another example of a sensor chip formeasuring a Hct value of the present invention.

FIG. 30 is an exploded perspective view showing an example of a sensorchip for measuring an analyte concentration of the present invention.

FIG. 31 is a plan view showing an example of a sensor chip for measuringan analyte concentration of the present invention.

FIG. 32 is an exploded perspective view showing another example of asensor chip for measuring an analyte concentration of the presentinvention.

FIG. 33 is a plan view showing another example of a sensor chip formeasuring an analyte concentration of the present invention.

FIG. 34 is an exploded perspective view showing another example of asensor chip for measuring an analyte concentration of the presentinvention.

FIG. 35 is a plan view showing another example of a sensor chip formeasuring an analyte concentration of the present invention.

FIG. 36 is a diagram showing an example of a circuit structure of asensor unit for measuring an analyte concentration of the presentinvention.

FIG. 37 is an exploded perspective view showing another example of asensor chip for measuring an analyte concentration of the presentinvention.

FIG. 38 is a plan view showing another example of a sensor chip formeasuring an analyte concentration of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

In a measurement of Hct value by the present invention, the contactpattern of a redox substance and electrodes is controlled such that theoxidant of the redox substance is substantially not in contact with aworking electrode but in contact with a counter electrode, and that thereductant of the redox substance is substantially not in contact withthe counter electrode but is in contact with the working electrode.

The oxidant or the reductant of the redox substance being in contactwith the counter electrode or working electrode may be a state in which,for example, a blood sample containing the redox substance is in contactwith the electrodes, or a state in which, for example, the redoxsubstance is disposed on the electrodes or embedded in the surfaces ofthe electrodes. That is, in the measurement of Hct value, the redoxsubstance may be in contact with the electrodes by being dissolved inthe blood sample, or by being provided as a solid.

The reductant is a substance that undergoes electrochemical oxidation atthe working electrode in response to an applied voltage of 3.0 V orless, when the working electrode is the anode and the counter electrodeis the cathode. Examples of the reductant include reductants ofreversible electroactive compounds such as ferrocyanides,p-hydroquinone, p-hydroquinone derivative, reduced phenazinemethosulfate, leucomethylene blue, ferrocene, and ferrocene derivative.Other examples are ascorbic acid, uric acid, acetaminophen, silver,copper, and nickel, among others. Preferably, the reductant is aferrocyanide, which is preferably potassium ferrocyanide.

The oxidant is a substance that undergoes electrochemical reduction atthe counter electrode in response to an applied voltage of 3.0 V orless, when the working electrode is the anode and the counter electrodeis the cathode. Examples of the oxidant include oxidants of reversibleelectroactive compounds such as ferricyanides, p-benzoquinone,p-benzoquinone derivative, oxidized phenazine methosulfate, methyleneblue, ferricinium, and ferricinium derivative. The oxidant is preferablya ferricyanide, which is preferably potassium ferricyanide.

The amount of redox substance in contact with the electrodes (wordingelectrode and counter electrode) in the measurement of Hct value may becontrolled by adding, for example, a 0.1 to 1000 mM, 1 to 500 mM, or insome cases, 10 to 200 mM oxidant and reductant to the blood samplebrought into contact with the electrodes.

In the measurement of Hct value, the redox substance intrinsicallycontained in the blood sample (for example, human blood) is disregardedas the redox substance in contact with the electrodes (working electrodeand counter electrode). In other words, in this specification, the redoxsubstance intrinsically contained in the blood sample is regarded asbeing substantially not in contact with the electrodes. Further, in thisspecification, a blood sample containing the redox substance in anamount comparable to that intrinsically contained in human blood isregarded as being substantially not containing the redox substance.

The oxidant or the reductant substantially not being in contact with theelectrodes may be a state in which the surface of the electrode iscovered with a reagent substantially not containing the oxidant or thereductant. For example, a reagent substantially not containing thereductant may be disposed to cover the surface of the counter electrodeto realize a state in which the reductant is substantially not incontact with the counter electrode, even when the blood sample containsthe reductant.

The material of the working electrode is preferably a conductingmaterial, for example, such as palladium, platinum, gold, titanium, orcarbon, that does not readily undergo oxidation when a voltage of 3.0 Vor less is applied to the working electrode, when the working electrodeis the anode and the counter electrode is the cathode. With the workingelectrode made of such a conducting material, the Hct value of the bloodsample can be measured more stably. The working electrode may be, forexample, an electrode core made of a conducting material exemplifiedabove, or may include, for example, a polymer film formed on theelectrode core. Examples of the material of the polymer film include:carboxymethyl cellulose; hydroxyethyl cellulose; hydroxypropylcellulose; methyl cellulose; ethyl cellulose; ethylhydroxyethylcellulose; carboxyethyl cellulose; polyvinyl alcohol; polyvinylpyrrolidone; polyamino acid such as polylysine; polystyrene sulfonate;gelatin and a derivative thereof polyacrylic acid and a salt thereof,polymethacrylic acid and a salt thereof starch and a derivative thereofmaleic anhydride polymer and a salt thereof and agarose gel and aderivative thereof. These compounds may be used either individually orin a combination of two or more kinds.

A conducting material used for the counter electrode is not particularlylimited. The counter electrode may be, for example, an electrode coremade of a known conducting material as represented by the examples givenabove, or may include, for example, the polymer film formed on theelectrode core.

The shape and size of the working electrode and counter electrode arenot particularly limited. The layout pattern of the working electrodeand counter electrode on an insulating substrate is not particularlylimited either. However, a stable measurement of Hct value in a bloodsample would be possible when the closest distance between the workingelectrode and the counter electrode is 0.05 mm or greater, 0.1 mm orgreater, or in some cases, 0.5 mm or greater. The upper limit of theclosest distance is not particularly limited.

In the measurement of Hct value, a voltage of 3.0 V or less is appliedacross the working electrode and the counter electrode (Hct valuemeasuring voltage), when the working electrode is the anode and thecounter electrode is the cathode. In the present invention, theapplication of the Hct value measuring voltage across the workingelectrode and the counter electrode as the anode and the cathode,respectively, stably can produce a current associated with the oxidationof the reductant in contact with the working electrode and the reductionof the oxidant in contact with the counter electrode, immediately afterthe voltage application, even when the applied voltage is 3.0 V or less,or even 1.0 V or less. Though the reason for this is unclear, it appearsto be due to the oxidation current that occurs solely by the oxidationreaction of the reductant in contact with the working electrode, and thegradual formation of an oxide film on the surface of the workingelectrode, in the measurement of Hct value.

The Hct value measuring voltage is applied for, for example, 0.001 to 60seconds, preferably 0.01 to 10 seconds, more preferably 0.01 to 5seconds, and even more preferably 0.01 to 3 seconds. The Hct valuemeasuring voltage may be, for example, 0.75 V or less, 0.5 V or less,0.25 V or less, 0.15 V or less, or 0.1 V or less, when the workingelectrode is the anode and the counter electrode is the cathode. Thelower limit of the Hct value measuring voltage is not particularlylimited as long as the reductant is oxidized at the working electrodeand the oxidant is reduced at the counter electrode. However, it isdesirable that the Hct value measuring voltage exceed 0 V and create apositive potential at the working electrode, when the working electrodeis the anode and the counter electrode is the cathode.

The Hct value of the blood sample is calculated based on the currentthat flows between the working electrode and the counter electrode bythe application of the Hct value measuring voltage. The Hct value can becalculated, for example, by referring to a standard curve or a standardtable relating Hct value to the amount of current after a predeterminedtime period from the application of the Hct value measuring voltage.

The Hct value described above can be measured using a sensor chip formeasuring a Hct value, which is an example of a sensor chip of thepresent invention.

The sensor chip for measuring a Hct value includes a Hct value analyzer,which electrochemically detects a current reflecting the Hct value ofthe blood sample. The Hct value analyzer includes an electrode systemhaving the working electrode and the counter electrode, and a bloodsample holder used to hold a blood sample in contact with the workingelectrode and the counter electrode. The blood sample holder is incommunication with a blood sample inlet through which a blood sample isintroduced.

The working electrode and the counter electrode are disposed to at leastpartially face the blood sample holder, such that the working electrodeand the counter electrode are in contact with the blood sampleintroduced into the blood sample holder. The surfaces of the workingelectrode and the counter electrode facing the blood sample holder maybe, for example, the surfaces of the electrode cores forming theseelectrodes, or, for example, the surfaces of the polymer films formed onthe electrode cores.

The electrode cores of the working electrode and the counter electrodecan be formed, for example, by a screen printing method, a sputteringmethod, or a vapor-deposition method. The polymer film can be formed,for example, from a solution of polymer material for forming the film,by applying the solution on the electrode core and drying it. The shapeand size of the working electrode and counter electrode, and the layoutpattern of these electrodes on the insulating substrate are notparticularly limited. However, the Hct value of the blood sample stablycan be measured more easily when the closest distance between theworking electrode and the counter electrode falls in the rangesexemplified above.

In the Hct value analyzer, when a blood sample is introduced into theblood sample holder and a voltage is applied across the workingelectrode and the counter electrode, the blood sample should be incontact with the counter electrode and the working electrode such thatthe oxidant of the redox substance is substantially not in contact withthe working electrode but in contact with the counter electrode, andthat the reductant of the redox substance is substantially not incontact with the counter electrode but in contact with the workingelectrode. To this end, the layout pattern of the reagents containingthe oxidant or the reductant of the redox substance, the shape of theblood sample holder, and the relative layout pattern of the counterelectrode and the working electrode are set as follows.

(1) A first reagent containing the oxidant of the redox substance andsubstantially not containing the reductant of the redox substance isdisposed to cover the surface of the counter electrode facing the bloodsample holder, and a second reagent containing the reductant andsubstantially not containing the oxidant is disposed to cover thesurface of the working electrode facing the blood sample holder. Withthis layout of the reagents covering the surfaces of the respectiveelectrodes, the surface of each electrode will be in contact with onlythe intended form, either the oxidant or the reductant, of the redoxsubstance and substantially not in contact with the redox substance ofthe other form in the measurement of Hct value, even when the redoxsubstance in one of the reagents dissolves into the introduced bloodsample and migrates with the blood sample to the area near the electrodeon which the other reagent is disposed.

(2) When the blood sample holder includes an inlet portion incommunication with the blood sample inlet, and a first and a secondbranch portion branching out of the inlet portion, and when the firstbranch portion faces the counter electrode and the second branch portionfaces the working electrode, the first reagent and the second reagentmay be disposed on the first branch portion and the second branchportion, respectively. With this layout, the surface of each electrodewill be in contact with only the intended oxidant or reductant, andsubstantially not in contact with the redox substance of the other formas in the case of (1). At the branch portion, the reagent may beseparated from the electrode, or in contact with the electrode.

(3) In the blood sample holder, one of the first reagent and the secondreagent is disposed on the upstream side of the other reagent withrespect to the flow of the blood sample introduced into the blood sampleholder through the blood sample inlet. (a) When the other reagent is thefirst reagent, the counter electrode may be disposed on the downstreamside of the working electrode with respect to the flow of the bloodsample, and the first reagent may be disposed to cover the surface ofthe counter electrode facing the blood sample holder. The workingelectrode may be disposed on the upstream side of the counter electrodewith respect to the flow of the blood sample, and the second reagent maybe disposed separately from the working electrode on the upstream sideof the working electrode with respect to the flow of the blood sample,or may be disposed in contact with the working electrode. (b) When theother reagent is the second reagent, the working electrode may bedisposed on the downstream side of the counter electrode with respect tothe flow of the blood sample, and the second reagent may be disposed tocover the surface of the working electrode facing the blood sampleholder. The counter electrode may be disposed on the upstream side ofthe working electrode with respect to the flow of the blood sample, andthe second reagent may be disposed separately from the counter electrodeon the upstream side of the counter electrode with respect to the flowof the blood sample, or may be disposed in contact with the counterelectrode. With this layout of the reagent covering the surface of theelectrode disposed on the downstream side, the surface of each electrodewill be in contact with only the intended form, either the oxidant orthe reductant, of the redox substance and substantially not in contactwith the redox substance of the other form in the measurement of Hctvalue, even when the redox substance in one of the reagents disposed onthe upstream side of the downstream electrode dissolves into theintroduced blood sample and migrates with the blood sample to the areanear the downstream electrode on which the other reagent is disposed.

The reagent containing the redox substance (oxidant, reductant) furthermay include other compounds. Some of the examples of such additionalcompounds include: amino acids (homogenizer) such as taurine, glycine,serine, proline, threonine, and lycine; carboxymethyl cellulose;hydroxyethyl cellulose; hydroxypropyl cellulose; methyl cellulose; ethylcellulose; ethylhydroxyethyl cellulose; carboxyethyl cellulose;polyvinyl alcohol; polyvinyl pyrrolidone; polyamino acid such aspolylysine; polystyrene sulfonate; gelatin and a derivative thereof,polyacrylic acid and a salt thereof, polymethacrylic acid and a saltthereof, starch and a derivative thereof, maleic anhydride polymer and asalt thereof, and agarose gel. The amount of redox substance disposed inthe Hct value analyzer may be set such that the amount of redoxsubstance in contact with the electrodes in the measurement of Hct valueis, for example, in a concentration of 0.1 to 1000 mM, 1 to 500 mM, orin some cases, 10 to 200 mM.

The shape and volume of the blood sample holder desirably are set suchthat the blood sample can be introduced therein by capillary action.

FIGS. 1 through 6, and FIGS. 28 and 29 are diagrams depicting specificexamples of the layout pattern of the reagents containing the redoxsubstance, the shape of the blood sample holder, and the relative layoutpattern of the counter electrode and the working electrode, in thesensor chip for measuring a Hct value.

<Sensor Chip A for Measuring Hct Value>

FIG. 1 is an exploded perspective view of a sensor chip A for measuringa Hct value, and FIG. 2 is a plan view of the sensor chip shown inFIG. 1. As shown in the figures, a sensor chip A100 a for measuring aHct value includes a spacer 102 having a T-shaped cutout portion 104, aninsulating substrate 101, and a cover 103. The cover 103 is disposed onthe insulating substrate 101 with the spacer 102 in between, leaving oneend portion of the insulating substrate 101 uncovered (on the right inthe figures). These members 101, 102, and 103 are integrated, forexample, by bonding or heat fusion. The cutout portion 104 of the spacer102 serves as a blood sample holder 14 after integration of the members.The blood sample holder 14 includes an inlet portion 17 extending alongthe longer side of the chip 100 a, and two branch portions 18 a and 18 bbranching out of the inlet portion 17 and extending along the shorterside of the chip 100 a.

The inlet portion 17 is in communication with the outside at an endportion of the spacer 102 (on the left in the figures). In other words,the blood sample holder 14 is in communication with a blood sample inlet16 that opens to the outside of the chip 100 a. The cover 103 includesoutlets 15, respectively corresponding in position to the ends of thebranch portions 18 a and 18 b. A working electrode 11 and a counterelectrode 12 are disposed on the insulating substrate 101 such that aportion (portion 31) of the working electrode 11 and a portion (portion32) of the counter electrode 12 face the branch portions 18 a and 18 b,respectively. The working electrode 11 and the counter electrode 12 eachare connected to a lead (not shown). An end of each lead is exposed tooutside of the chip 100 a at the end portion of the insulating substrate101 not covered with the spacer 102 and the cover 103, in order to applya voltage across the working electrode and the counter electrode.

A first reagent 13 containing the oxidant of the redox substance andsubstantially not containing the reductant is disposed in contact withthe portion 32 of the counter electrode 12. A second reagent 19containing the reductant of the redox substance and substantially notcontaining the oxidant is disposed in contact with the portion 31 of theworking electrode 11. These reagents may not easily dissolve into theblood sample or may dissolve easily into the blood sample.

Preferably, the first reagent 13 is disposed in contact with only theportion 32 of the counter electrode in the blood sample holder 14. Bydisposing the reagent this way, a pure blood sample substantially notcontaining the oxidant of the redox substance can be placed in a largequantity between the working electrode and the counter electrode in themeasurement of Hct value. This improves the detection sensitivity of theHct value.

The materials of the insulating substrate, the spacer, and the cover arenot particularly limited as long as the working electrode and thecounter electrode are not shorted by the integration. Examples of suchmaterials include polyethylene terephthalate (PET), polycarbonate (PC),polyimide (PI), polyethylene (PE), polypropylene (PP), polystyrene (PS),polyvinyl chloride (PVC), polyoxymethylene (POM), monomer-cast nylon(MC), polybutylene terephthalate (PBT), methacrylic resin (PMMA), ABSresin (ABS), and glass.

In a sensor chip for measuring a Hct value of the present invention, oneof the requirements in introducing a blood sample into the blood sampleholder and applying a voltage across the working electrode and thecounter electrode is that the blood sample is in contact with thecounter electrode and the working electrode while the oxidant of theredox substance from the reagent disposed in the sensor chip is incontact with the counter electrode but substantially not in contact withthe working electrode, and the reductant of the redox substance from thereagent disposed in the sensor chip is in contact with the workingelectrode but substantially not in contact with the counter electrode.So long as these conditions are met, any arrangement can be madeconcerning the layout pattern of the first and second reagents in theblood sample holder, the shape of the blood sample holder, the layoutpattern of the reagents containing the redox substance, and the relativelayout pattern of the counter electrode and the working electrode. Otherexemplary configurations of a sensor chip for measuring a Hct value ofthe present invention are described below.

<Sensor Chip B for Measuring Hct Value>

FIG. 28 is an exploded perspective view of a sensor chip B for measuringa Hct value, and FIG. 29 is a plan view of the sensor chip shown in FIG.28. As shown in the figures, a sensor chip B100 b for measuring a Hctvalue has the same configuration as the sensor chip A for measuring aHct value except that, at the branch portion 18 a, the second reagent 19is separated from the portion 31 of the working electrode 11 and iscloser to the inlet portion 17 than the portion 31 is, and that, at thebranch portion 18 b, the first reagent 13 is separated from the portion32 of the counter electrode 12 and is closer to the inlet portion 17than the portion 32 is. The first reagent and the second reagent easilydissolve into the blood sample introduced into the branch portions 18 aand 18 b.

<Sensor Chip C for Measuring Hct Value>

FIG. 3 is an exploded perspective view of a sensor chip C for measuringa Hct value, and FIG. 4 is a plan view of the sensor chip shown in FIG.3. As shown in the figures, a sensor chip C100 c for measuring a Hctvalue includes a spacer 202 having a rectangular cutout portion 204, andinsulating substrate 201, and a cover 203. The cover 203 is disposed onthe insulating substrate 201 with the spacer 202 in between, leaving oneend portion of the insulating substrate 201 uncovered (on the right inthe figures). These members 201, 202, and 203 are integrated, forexample, by bonding or heat fusion. The cutout portion 204 of the spacer202 serves as a blood sample holder 24 after integration of the members.The blood sample holder 24 extends along the longer side of the chip 100c, and is in communication with outside at an end portion of the spacer202 (on the left in the figures). In other words, the blood sampleholder 24 is in communication with a blood sample inlet 26, which opensto outside of the chip 100 c. The cover 203 includes an outlet 25,corresponding in position to a portion of the blood sample holder 24 atthe opposite end of the end in communication with outside. A workingelectrode 21 and a counter electrode 22 are disposed on the insulatingsubstrate 201 such that a portion (portion 41) of the working electrode21 and a portion (portion 42) of the counter electrode 22 face the bloodsample holder 24, and that the portion 41 is closer to a blood sampleinlet 26 than the portion 42 is. The working electrode 21 and thecounter electrode 22 each are connected to a lead (not shown). An end ofeach lead is exposed to outside of the chip 100 c at the end portion ofthe insulating substrate 201 not covered with the spacer 202 and thecover 203, in order to apply a voltage across the working electrode andthe counter electrode.

A first reagent 23 containing the oxidant of the redox substance andsubstantially not containing the reductant is disposed to cover theportion 42 of the counter electrode 22. A second reagent 27 containingthe reductant of the redox substance and substantially not containingthe oxidant is disposed to cover the portion 41 of the working electrode21. Preferably, these reagents do not easily dissolve into the bloodsample.

<Sensor Chip D for Measuring Hct value>

FIG. 5 is an exploded perspective view of a sensor chip D for measuringa Hct value, and FIG. 6 is a plan view of the sensor chip shown in FIG.5. As shown in the figures, a sensor chip D100 d for measuring a Hctvalue has the same configuration as the sensor chip C for measuring aHct value except that the counter electrode 22 and the working electrode21 are disposed on the insulating substrate 201 such that the portion 42of the counter electrode 22 is closer to the blood sample inlet 26 thanthe portion 41 of the working electrode 21 is, and that the firstreagent 23 and the second reagent 27 are disposed to cover the portion42 of the counter electrode 22 and the portion 41 of the workingelectrode 21, respectively. Preferably, these reagents do not easilydissolve into the blood sample.

The measurement of the Hct value of the blood sample by the sensor chipfor measuring a Hct value can be performed using, for example, a sensorunit for measuring a Hct value, which is an example of a sensor unit ofthe present invention.

The sensor unit for measuring a Hct value includes a sensor chip formeasuring a Hct value, and a sensor main body detachably provided withthe sensor chip. The sensor main body includes a voltage applyingcircuit capable of applying a predetermined voltage across the workingelectrode and the counter electrode of the sensor chip with the sensorchip attached to the sensor main body.

The voltage applying circuit applies a voltage of 3.0 V or less acrossthe working electrode and the counter electrode, when the workingelectrode of the anode and the counter electrode is the cathode. Theapplied voltage may be, for example, 1.0 V or less, 0.75 V or less, 0.5V or less, 0.25 V or less, 0.15 V or less, or 0.1 V or less. The lowerlimit of the voltage is not particularly limited as long as thereductant is oxidized at the working electrode and the oxidant isreduced at the counter electrode. However, the voltage is desirably 0 Vor greater, when the working electrode is the anode and the counterelectrode is the cathode.

FIG. 26 is a diagram showing an example of the sensor unit for measuringa Hct value. A sensor unit 126 for measuring a Hct value includes a flathexahedral sensor main body 123, and a sensor chip 121 for measuring aHct value. Through one side wall surface of the sensor main body 123, anattachment opening 125 is provided in the shape of a rectangularaperture. The sensor chip 121 is attached to the sensor main body 123 bybeing detachably coupled to the attachment opening 125. A displaysection 124 for displaying a measurement result of Hct value is providedsubstantially at the center of one principal surface of the sensor mainbody 123.

FIG. 27 is a diagram showing an exemplary circuit structure formeasuring a Hct value in the sensor unit 126 for measuring a Hct value.The sensor main body 123 includes a voltage applying circuit 110 forapplying a predetermined voltage across the working electrode 11 and thecounter electrode 12 of the sensor chip 121, and a liquid crystaldisplay (LCD) 115 as the display section 124. The voltage applyingcircuit 110 includes two connectors 111 a and 111 b, a current/voltageconverting circuit 112, an A/D converting circuit 113, a centralprocessing unit (CPU) 114, and a reference voltage source 116. Theseelements 111 a, 111 b, 112, 113, 114, 115, and 116 are connectedelectrically to one another, as indicated by the solid lines in FIG. 27.

The measurement of the Hct value of the blood sample using the sensorunit 126 proceeds as follows, for example. First, a blood sample isintroduced into the blood sample holder 14 of the sensor chip 121through a blood sample inlet 122 of the sensor chip 121. Then, under aninstruction from the CPU 114, a predetermined Hct value measuringvoltage is applied across the working electrode 11 and the counterelectrode 12 by the current/voltage converting circuit 112 and thereference voltage source 116. The Hct value measuring voltage is appliedfor an adjusted time period in the foregoing range, for example, 0.001to 60 seconds, preferably 0.01 to 10 seconds, more preferably 0.01 to 5seconds, and even more preferably 0.01 to 3 seconds. The value of thecurrent flown between the working electrode 11 and the counter electrode12 by the application of the Hct value measuring voltage is converted toa voltage value by the current/voltage converting circuit 112. Thisvoltage value is then converted to a digital value by the A/D convertingcircuit 113 before it is sent to the CPU 114. The CPU 114 calculates aHct value based on the digital value. The Hct value is calculated, forexample, by referring to a standard curve or a standard table, relatingthe Hct value of a blood sample to the amount of current after apredetermined time period from the application of the Hct valuemeasuring voltage. The result of calculation is visually displayed onthe LCD 115.

With a method for measuring a Hct value of a blood sample of the presentinvention, the analyte concentration in the blood sample can be measuredwith improved accuracy. The analyte concentration in the blood sample isdetermined based on data C, which is obtained by correcting preliminarymeasurement data (data B) of the analyte in the blood sample using dataA, which corresponds to the Hct value of the blood sample.

The data A corresponding to the Hct value of the blood sample isobtained by a method for measuring a Hct value of a blood sample of thepresent invention. The data A may be a value that results from theconversion of current A into a Hct value, wherein the current A is acurrent flowing through the working and counter electrodes for Hct valuemeasurement (a working and a counter electrode for correction),reflecting the Hct value of the blood sample. Alternatively, the data Amay be a value obtained by the conversion of the current A into someother parameter different from the Hct value. Further, the data A may bethe current A itself. The conversion of the current A into a Hct valueis performed, for example, by referring to a standard curve or astandard table, relating current A to Hct value after a predeterminedtime period from the application of the Hct value measuring voltage.

Current B is detected to obtain data B. The current B is a currentflowing between a working electrode (working electrode for preliminarymeasurement) and a counter electrode (counter electrode for preliminarymeasurement), as a result of applying a voltage (preliminary measuringvoltage) across these electrodes in contact with a blood sample after acertain time period of reaction between the analyte in the blood sampleand a redox enzyme that uses the analyte as a substrate. The data B maybe, for example, a value that results from the conversion of the currentB into a preliminary measurement concentration of the analyte.Alternatively, the data B may be, for example, a value obtained by theconversion of the current B into some other parameter different from thepreliminary measurement concentration. Further, the data B may be thecurrent B itself, for example. The conversion of the current B into apreliminary measurement concentration is performed, for example, byreferring to a standard curve or a standard table, relating current B topreliminary measurement concentration after a predetermined time periodfrom the application of the preliminary measuring voltage.

The current B is detected using a redox substance, for example, areversible electroactive compound as represented by ferricyanide, whichmediates the movement of electrons between the enzyme reaction and theelectrode reaction. The content of the redox substance in the bloodsample brought into contact with the working electrode for preliminarymeasurement and the counter electrode for preliminary measurement may be0.1 to 1000 mM, for example. In the detection of current B, the redoxenzyme and the redox substance may be in contact with the counterelectrode for preliminary measurement and the working electrode forpreliminary measurement by being contained in the blood sample broughtinto contact with these electrodes, for example. Alternatively, theredox enzyme and the redox substance directly may be disposed on theseelectrodes, for example. Further, the redox enzyme and the redoxsubstance may be embedded in the surfaces of these electrodes, forexample. That is, in the detection of current B, the redox enzyme andthe redox substance may be in contact with the electrodes by beingdissolved in the blood sample, or by being provided as a solid.

The analyte in the blood sample may be a substance other than bloodcells. Some of the examples include glucose, albumin, lactic acid,bilirubin, and cholesterol. The redox enzyme is selected according tothe type of substrate, i.e., the analyte being analyzed. Examples of theredox enzyme include glucose oxidase, glucose dehydrogenase, lactateoxidase, lactate dehydrogenase, bilirubin oxidase, and cholesteroloxidase. The amount of redox enzyme that reacts with the analyte may beset such that the content of the redox enzyme in the blood sample is,for example, 0.01 to 100 units (U), 0.05 to 10 U, or in some cases, 0.1to 5 U.

The reaction time of the analyte and the redox enzyme may be, forexample, 0 to 60 seconds, 0.5 to 30 seconds, or in some cases, 1 to 10seconds. The preliminary measuring voltage may be, for example, 0.05 to1 V, 0.1 to 0.8 V, or in some cases, 0.2 to 0.5 V, when the workingelectrode for preliminary measurement is the anode and the counterelectrode for preliminary measurement is the cathode. The preliminarymeasuring voltage may be applied for, for example, 0.01 to 30 seconds,0.1 to 10 seconds, or in some cases, 1 to 5 seconds.

The counter electrode for preliminary measurement or the workingelectrode for preliminary measurement may be provided separately fromthe counter electrode for correction or the working electrode forcorrection. Alternatively, part of or all of the counter electrode forcorrection or the working electrode for correction may be used as thecounter electrode for preliminary measurement or the working electrodefor preliminary measurement. For example, the working electrode forpreliminary measurement also may be used as the counter electrode forcorrection.

The counter electrode for preliminary measurement and the workingelectrode for preliminary measurement can be configured in the samemanner as the counter electrode for correction. The shape, size, andlayout pattern of the counter electrode for preliminary measurement andthe working electrode for preliminary measurement are not particularlylimited.

The order of detecting the current A and current B is not particularlylimited. For example, when the working electrode for preliminarymeasurement and the counter electrode for correction are realized by asingle electrode as mentioned above, it is preferable that the current Abe detected after detecting the current B, considering the possibleshortage of the redox substance of the form brought into contact withthe electrode in the detection of each current. This needs to beprevented because the redox reaction on the electrode becomes arate-limiting step in this case.

As described, the analyte concentration in the blood sample isdetermined based on data C, which is obtained by correcting data B withdata A. The resulting value of data C corresponds to data B. The data Cmay be, for example, the analyte concentration itself in the bloodsample, or a corrected current value. When the value of data C is notthe analyte concentration itself, the analyte concentration in the bloodsample is determined by referring to a standard curve or a standardtable, relating the value of data C to the analyte concentration in theblood sample.

The analyte concentration in the blood sample can be measured using asensor chip for measuring an analyte concentration, which is anotherexample of a sensor chip of the present invention.

The sensor chip for measuring an analyte concentration includes a Hctvalue analyzer, analogous to that in the sensor chip for measuring a Hctvalue.

The sensor chip for measuring an analyte concentration includes ananalyzer for preliminary measurement, used for electrochemical detectionof the current B. The analyzer for preliminary measurement may beprovided separately from the Hct value analyzer, or part of or all ofthe Hct value analyzer may be used as the analyzer for preliminarymeasurement. For example, the electrode system (electrode system A), theblood sample holder (blood sample holder A), and the blood sample inlet(blood sample inlet A) of the Hct value analyzer may be used to realizean electrode system (electrode system B) including the working electrodefor preliminary measurement and the counter electrode for preliminarymeasurement, a blood sample holder (blood sample holder B) for holdingthe blood sample in contact with the working electrode for preliminarymeasurement and the counter electrode for preliminary measurement, and ablood sample inlet (blood sample inlet B) in communication with theblood sample holder B, respectively.

When the analyzer for preliminary measurement is separately providedfrom the Hct value analyzer, the blood sample inlet B of the analyzerfor preliminary measurement may be provided more toward the downstreamside compared to the Hct value analyzer, with respect to the flow of theblood sample introduced into the sensor chip, so that, in the detectionof current A, the oxidant of the redox substance will not be in contactwith the working electrode for correction as a result of the inflow ofthe blood sample. When part of or all of the Hct value analyzer is usedas the analyzer for preliminary measurement, the layout pattern of thereagent containing the oxidant, the shape of the blood sample holder,and the layout pattern of each electrode system may be set in the mannerdescribed later. Note that, when at least part of the electrode system Ais used to realize the electrode system B, the working electrode forpreliminary measurement and the counter electrode for correction may berealized by a single electrode, as described above.

The working electrode for preliminary measurement and the counterelectrode for preliminary measurement at least partially face the bloodsample holder B, so as to be in contact with the blood sample introducedinto the blood sample holder B.

Desirably, the shape and volume of the blood sample holder B are setsuch that the blood sample can be introduced therein by capillaryaction.

The analyzer for preliminary measurement may include the redox enzymeand the redox substance associated with the enzymatic cycling reactionfor the preliminary measurement of the analyte concentration. The redoxenzyme may contain an enzyme stabilizer as represented by, for example,a sugar alcohol such as maltitol, sorbitol, and xylitol. The amount ofredox enzyme in the analyzer for preliminary measurement may be set suchthat the content of the redox enzyme in the blood sample is, forexample, 0.01 to 100 units (U), 0.05 to 10 U, or in some cases, 0.1 to 5U.

FIGS. 30 through 35, and FIGS. 37 and 38 are diagrams depicting specificexamples of the layout pattern of the reagent containing the oxidant,the shape of the blood sample holder, and the layout pattern of theelectrode system in the sensor chip for measuring an analyteconcentration. In all of these examples, the analyzer for preliminarymeasurement and the Hct value analyzer share some of the samecomponents. Specifically, the working electrode for preliminarymeasurement and the counter electrode for correction are realized by asingle electrode, and the blood sample holder A and the blood sampleinlet A also serve as the blood sample holder B and the blood sampleinlet B, respectively.

<Sensor Chip A for Measuring Analyte Concentration>

FIG. 30 is an exploded perspective view of a sensor chip A for measuringan analyte concentration, and FIG. 31 is a plan view of the sensor chipshown in FIG. 30. As shown in the figures, a sensor chip A200 a formeasuring an analyte concentration has the same configuration as thesensor chip A100 a for measuring a Hct value except that a counterelectrode 30 for preliminary measurement is disposed on the insulatingsubstrate 101 such that a portion (portion 33) of the counter electrode30 for preliminary measurement faces the branch portion 18 b and iscloser to the inlet portion 17 than the portion 32 is. The counterelectrode 12 also serves as the working electrode for preliminarymeasurement. The counter electrode 30 for preliminary measurement isconnected to a lead (not shown). An end of the lead is exposed to theoutside of the chip 200 a at the end portion of the insulating substrate101 not covered with the spacer 102 and the cover 103.

Another electrode may be disposed on the insulating substrate. Forexample, a blood detecting electrode for detecting an inflow of asufficient measurement amount of blood sample into the blood sampleholder may be disposed on the insulating substrate such that a portionof the blood detecting electrode faces the blood sample holder and isfarther from the blood sample inlet than the portion 33 is.

<Sensor Chip B for Measuring Analyte Concentration>

FIG. 37 is an exploded perspective view of a sensor chip B for measuringan analyte concentration, and FIG. 38 is a plan view of the sensor chipshown in FIG. 37. As shown in the figures, a sensor chip B200 b formeasuring an analyte concentration has the same configuration as thesensor chip A for measuring an analyte concentration except that, in thebranch portion 18 a, the second reagent 19 is separated from the portion31 of the working electrode 11 and is closer to the inlet portion 17than the portion 31 is, and that, in the branch portion 18 b, the firstreagent 13 is separated from the portion 32 of the counter electrode 12and is closer to the inlet portion 17 than the portion 32 is.

<Sensor Chip C for Measuring an Analyte Concentration>

FIG. 32 is an exploded perspective view of a sensor chip C for measuringan analyte concentration, and FIG. 33 is a plan view of the sensor chipshown in FIG. 32. As shown in the figures, a sensor chip C200 c formeasuring an analyte concentration has the same configuration as thesensor chip C100 c for measuring a Hct value except that a counterelectrode 28 for preliminary measurement, having a branched U-shapedportion (portion 43) facing the blood sample holder 24 and extending onthe both sides of the portion 42 is disposed on the insulating substrate201. The counter electrode 22 also serves as the working electrode forpreliminary measurement. The counter electrode 28 for preliminarymeasurement is connected to a lead (not shown). An end of the lead isexposed to outside of the chip 200 c at the end portion of theinsulating substrate 201 not covered with the spacer 202 and the cover203.

<Sensor Chip D for Measuring Analyte Concentration>

FIG. 34 is an exploded perspective view of a sensor chip D for measuringan analyte concentration, and FIG. 35 is a plan view of the sensor chipshown in FIG. 34. As shown in the figures, a sensor chip D200 d formeasuring an analyte concentration has the same configuration as thesensor chip C for measuring an analyte concentration except that thecounter electrode 22 and the working electrode 21 are disposed on theinsulating substrate 201 such that the portion 42 of the counterelectrode 22 is closer to the blood sample inlet 26 than the portion 41of the working electrode 21 is, and that the first reagent 23 and thesecond reagent 27 are disposed to cover the portion 42 of the counterelectrode 22 and the portion 41 of the working electrode 21,respectively.

The measurement of the analyte concentration in the blood sample by thesensor chip for measuring an analyte concentration can be performedusing, for example, a sensor unit for measuring an analyteconcentration, which is another example of a sensor unit of the presentinvention.

The sensor unit for measuring an analyte concentration includes a sensorchip for measuring an analyte concentration, and a sensor main bodydetachably provided with the sensor chip. The sensor main body has thesame configuration as the sensor main body of the sensor unit formeasuring a Hct value shown in FIG. 26 except that a circuit for thepreliminary measurement of the analyte concentration in the blood sampleis provided in addition to the circuit for measuring a Hct value.

FIG. 36 is a diagram showing an exemplary circuit structure formeasuring an analyte concentration in a blood sample, in the sensor unitfor measuring an analyte concentration. A sensor main body 223 includes:a voltage applying circuit 210 for applying a voltage across at leasttwo of the electrodes selected from the working electrode 21 forcorrection, the counter electrode 22 for correction, the counterelectrode 28 for preliminary measurement, and the blood sample detectingelectrode 29 in the sensor chip 221 for measuring an analyteconcentration; and a liquid crystal display (LCD) 132 as a displaysection of the sensor main body. The voltage applying circuit 210 iscapable of applying a predetermined voltage across the working electrode21 for correction and the counter electrode 22 for correction, andswitching the applied potential to the electrodes so that the electrodecan be used as the anode or cathode. By the switching, the counterelectrode 22 for correction also can serve as the working electrode forpreliminary measurement. The voltage applying circuit 210 includes fourconnectors 137 a, 137 b, 137 c, and 137 d, a switching circuit 136, acurrent/voltage converting circuit 135, an A/D converting circuit 134, areference voltage source 133, and a central processing unit (CPU) 131.These elements 131, 132, 133, 134, 135, 136, 137 a, 137 b, 137 c, and137 d are connected electrically to one another, as indicated by thesolid lines in FIG. 36.

The measurement of the analyte concentration in the blood sample usingthe sensor unit for measuring an analyte concentration is performed asfollows, for example.

First, under an instruction from the CPU 131, the working electrode 21for correction is connected to the current/voltage converting circuit135 via the connector 137 d, and the blood sample detecting electrode 29is connected to the reference voltage source 133 via the connector 137b. This is followed by application of a certain voltage across theelectrodes under an instruction from the CPU 131. The applied voltagemay be, for example, 0.05 V to 1 V, when the working electrode forcorrection is the anode and the blood sample detecting electrode is thecathode. Introducing a blood sample into the blood sample holder 24 ofthe sensor chip 221 through the blood sample inlet of the sensor chip221 generates a current flow between the working electrode 21 forcorrection and the blood sample detecting electrode 29. The currentvalue is converted into a voltage value by the current/voltageconverting circuit 135, and is sent to the CPU 131 after conversion intoa digital value by the A/D converting circuit 134. Based on the digitalvalue, the CPU 131 detects the inflow of the blood sample into the bloodsample holder.

Following the inflow of the blood sample, the analyte in the bloodsample is allowed to react with the redox enzyme for, for example, 0 to60 seconds, so as to calculate a preliminary measurement concentrationof the analyte in the blood sample as follows. First, under aninstruction from the CPU 131, the switching circuit 136 comes intooperation to connect the counter electrode for preliminary measurement,also serving as the counter electrode 22 for correction, to thecurrent/voltage converting circuit 135 via the connector 137 a, and theworking electrode 28 for preliminary measurement to the referencevoltage source 133 via the connector 137 c. This is followed byapplication of a voltage of the foregoing range across the electrodes,under an instruction from the CPU 131. For example, when the workingelectrode for preliminary measurement is the anode and the counterelectrode for preliminary measurement is the cathode, a preliminarymeasuring voltage of 0.05 to 1 V is applied. The preliminary measuringvoltage is applied for an adjusted time period of, for example, 0.01 to30 seconds. The value of the current flowing between the electrodes bythe application of the preliminary measuring voltage is converted into avoltage value by the current/voltage converting circuit 135, and is sentto the CPU 131 after conversion into a digital value by the A/Dconverting circuit 134. Based on the digital value, the CPU 131calculates a preliminary measurement concentration of the analyte. Thepreliminary measurement concentration is calculated by referring to astandard curve or a standard table, relating the preliminary measurementconcentration of the analyte to the amount of current after apredetermined time period from the application of the preliminarymeasuring voltage.

After calculating the preliminary measurement concentration, the Hctvalue of the blood sample is calculated as follows, for example. First,under an instruction from the CPU 131, the switching circuit 136 comesinto operation to connect the working electrode 21 for correction to thecurrent/voltage converting circuit 135 via the connector 137 d, and thecounter electrode 22 for correction to the reference voltage source 133via the connector 137 a. This is followed by application of a Hct valuemeasuring voltage of 3.0 V or less across the electrodes under aninstruction from the CPU 131, when the working electrode for correctionis the anode and the counter electrode for correction is the cathode.The Hct value measuring voltage is applied for an adjusted time periodof, for example, 0.001 to 60 seconds. The value of the current flowingbetween the electrodes by the application of the Hct value measuringvoltage is converted into a voltage value by the current/voltageconverting circuit 135, and is sent to the CPU 131 after conversion intoa digital value by the A/D converting circuit 134. Based on the digitalvalue, the CPU 131 calculates an Hct value. The Hct value is calculatedby referring to, for example, a standard curve or a standard table,relating Hct value to the amount of current after a predetermined timeperiod from the application of the Hct value measuring voltage.

Then, in the CPU 131, the preliminary measurement concentrationcalculated as above is corrected based on the Hct value, so as todetermine the analyte concentration in the blood sample. The resultinganalyte concentration is displayed visually on the LCD 132. Thecorrection of the preliminary measurement concentration based on the Hctvalue is performed by referring to, for example, a standard curve or astandard table, relating the analyte concentration in the blood sampleto Hct value and preliminary measurement concentration.

The following will describe the present invention by way of examples andcomparative examples.

EXAMPLE 1

A sensor chip A for measuring a Hct value was prepared. Palladium wasused as the electrode cores of the working electrode and the counterelectrode. Using a spacer having a thickness of 100 μm, a 0.8 microliter(μL)-volume blood sample holder was formed. The effective areas of theworking electrode and the counter electrode in the blood sample holderwere 0.4 mm² and 0.7 mm², respectively, and the closest distance betweenthe working electrode and the counter electrode was 2.4 mm. A reactionreagent layer A containing the reductant but not the oxidant of theredox substance was disposed to cover the surface of the workingelectrode facing the blood sample holder. A reaction reagent layer Bcontaining the oxidant but not the reductant of the redox substance wasdisposed to cover the surface of the counter electrode facing the bloodsample holder. The reaction reagent layer A was disposed on the surfaceby applying a reagent solution, prepared by dissolving 50 mM potassiumferrocyanide (KANTO CHEMICAL CO., INC.) and 250 U/g of glucose oxidase(SIGMA) in a 0.5 mass % CMC aqueous solution (DAIICHI KOGYO CO., LTD.),on the surface of the electrode core of the working electrode (0.63mg/sensor), and then by drying the solution at 55° C. for 10 minutes.The reaction reagent layer B was disposed on the surface by applying areagent solution, prepared by dissolving 50 mM potassium ferricyanide(KANTO CHEMICAL CO., INC.) and 250 U/g of glucose oxidase in a 0.5 mass% CMC aqueous solution, on the surface of the electrode core of thecounter electrode (0.63 mg/sensor), and then by drying the solution at55° C. for 10 minutes. The cover had been rendered hydrophilic inadvance using a surfactant. The closest distance between the workingelectrode and the reaction reagent layer B was 1.8 mm.

Three kinds of blood samples with the Hct values of 25%, 45%, and 65%were prepared. Each blood sample was introduced into the blood sampleholder of the sensor chip, and a voltage of 3.0 V or less was appliedacross the working electrode and the counter electrode serving as theanode and the cathode, respectively. A resulting current (responsecurrent) flowing between the working electrode and the counter electrodewas measured. The results of measurement of response current arerepresented by the graphs shown in FIGS. 7 through 13. In each figure,graph (A) represents changes in response current value (μA) of eachblood sample as a function of time. Graph (B) represents changes inrelative amplitude values of the response currents obtained from the 25%and 65% Hct blood samples (sensitivity difference (%)) relative to theamplitude of the response current obtained from the 45% Hct bloodsample, as a function of time. In graph (A) and graph (B), thehorizontal axis represents time from the voltage application in seconds(sec).

As shown in the graphs, the sensor chip of Example 1 was able to detectresponse currents reflecting the Hct values of the blood samples with astable and distinct sensitivity difference, immediately after theapplication of a voltage of 3.0 V or less across the working electrodeand the counter electrode serving as the anode and the cathode,respectively.

EXAMPLE 2

A sensor chip was prepared as in Example 1 except that the reactionreagent layer A was prepared using a regent solution that had beenprepared by dissolving 50 mM potassium ferrocyanide and 1.0 mass %bovine serum albumin (SIGMA) in a 0.5 mass % CMC aqueous solution.

Each of the three kinds of blood samples was introduced into the bloodsample holder of the sensor chip, and a voltage of 3.0 V or less wasapplied across the working electrode and the counter electrode servingas the anode and the cathode, respectively. A resulting response currentflown between the working electrode and the counter electrode wasmeasured. The results of measurement of response current are representedby the graphs shown in FIGS. 14 through 19. As shown in the graphs, thesensor chip of Example 2 was able to detect response currents reflectingthe Hct values of the blood samples with a stable and distinctsensitivity difference, immediately after the application of a voltageof 3.0 V or less across the working electrode and the counter electrodeserving as the anode and the cathode, respectively.

COMPARATIVE EXAMPLE 1

A sensor chip was prepared as in Example 1, except that the reactionreagent layer A was not disposed.

Each of the three kinds of blood samples was introduced into the bloodsample holder of the sensor chip, and voltages of 2.0 V, 1.0 V, and 0.5V were applied across the working electrode and the counter electrodeserving as the anode and the cathode, respectively. A resulting responsecurrent flown between the working electrode and the counter electrodewas measured. The results of measurement of response current arerepresented by the graphs shown in FIGS. 20 through 22. As shown inFIGS. 21 and 22, a stable sensitivity difference was not obtained in thesensor chip of Comparative Example 1 when a voltage of 1.0 V or less wasapplied across the working electrode and the counter electrode servingas the anode and the cathode, respectively. More specifically, as shownin FIG. 21, when a voltage of 1.0 V was applied across the workingelectrode and the counter electrode serving as the anode and thecathode, respectively, the sensitivity difference fluctuated abruptlyimmediately after the voltage application, and, though the fluctuationsgradually leveled off, the sensitivity difference did not return to thenormal state after three seconds from the voltage application. Further,as shown in FIG. 22, when a voltage of 0.5 V was applied across theworking electrode and the counter electrode serving as the anode and thecathode, respectively, the sensitivity difference fluctuated abruptlyimmediately after the voltage application and continued fluctuating overa wide range. The sensitivity difference did not return to the normalstate after three seconds from the voltage application.

Though the reasons for these undesirable outcomes from the sensor chipof Comparative Example 1 are unclear, it appears that the results aredue to the redox current, generated by the electrolysis of water in theblood component, accounting for the majority of the redox current on theworking electrode.

COMPARATIVE EXAMPLE 2

A sensor chip was prepared that had the same configuration as the sensorchip C for measuring a Hct value, except that a reaction reagent layer Ccontaining the reductant and the oxidant was disposed to cover thesurface of each of the working electrode and the counter electrodefacing the blood sample holder, instead of the first reagent and thesecond reagent. Palladium was used as the electrode cores of the workingelectrode and the counter electrode. Using a spacer having a thicknessof 100 μm, a 304 nanoliter-volume blood sample holder was formed. Theeffective areas of the working electrode and the counter electrode inthe blood sample holder were 0.32 mm² and 0.4 mm², respectively, and theclosest distance between the working electrode and the counter electrodewas 0.05 mm. The reaction reagent layer C was disposed on the surface byapplying a reagent solution, prepared by dissolving 60 mM potassiumferricyanide, 8.25 mM potassium ferrocyanide, 1.0 mass % taurine(nacalai tesque), and 0.25 mass % maltitol (HAYASHIBARA) in a 0.1 mass %CMC aqueous solution, on the surface of the electrode core of eachelectrode (0.55 mg/sensor), and then by drying the solution at 20° C.for 50 minutes.

Each of the three kinds of blood samples was introduced into the bloodsample holder of the sensor chip, and a voltage of 0.2 V was appliedacross the working electrode and the counter electrode serving as theanode and the cathode, respectively. A resulting response currentflowing between the working electrode and the counter electrode wasmeasured. The results of measurement of response current are representedby the graph shown in FIG. 23. As shown in the graph, in the sensor chipof Comparative Example 2, the sensitivity difference was smallimmediately after the application of a voltage of 0.2 V across theworking electrode and the counter electrode serving as the anode and thecathode, respectively. Further, the sensitivity difference was unstablethroughout the measurement.

COMPARATIVE EXAMPLE 3

A sensor chip was prepared as in Comparative Example 2 except that areaction reagent layer D containing the oxidant but not the reductantwas disposed instead of the reaction reagent layer C. The reactionreagent layer D was disposed on the surface by applying a reagentsolution, prepared by dissolving 60 mM potassium ferricyanide, 1.0 mass% taurine, and 0.25 mass % maltitol in a 0.1 mass % CMC aqueoussolution, on the surface of the electrode core of each electrode (0.55mg/sensor), and then by drying the solution at 20° C. for 50 minutes.

Each of the three kinds of blood samples was introduced into the bloodsample holder of the sensor chip, and a voltage of 2.5 V was appliedacross the working electrode and the counter electrode serving as theanode and the cathode, respectively. A resulting response currentflowing between the working electrode and the counter electrode wasmeasured. The results of measurement of response current are representedby the graph shown in FIG. 24. As shown in the graph, in the sensor chipof Comparative Example 3, the sensitivity difference was smallimmediately after the voltage application, even with the application ofa relatively high voltage of 2.5 V across the working electrode and thecounter electrode serving as the anode and the cathode, respectively.Further, the sensitivity difference was unstable throughout themeasurement.

COMPARATIVE EXAMPLE 4

A sensor chip was prepared as in Comparative Example 2 except that:instead of the reaction reagent layer C, a reaction reagent layer Econtaining the reductant but not the oxidant was disposed to cover thesurface of the counter electrode facing the blood sample holder; neitherthe reaction reagent layer C nor the reaction reagent layer E wasdisposed to cover the surface of the working electrode facing the bloodsample holder; a CMC film was disposed on the surface of the electrodecore of the working electrode; and the effective areas of the workingelectrode and the counter electrode in the blood sample holder were 0.4mm² and 0.5 mm², respectively. The CMC film was disposed on the surfaceof the electrode core of the working electrode by dropping 0.01 to 100mg of 0.01 to 2.0 mass % CMC aqueous solution and then drying it. Thereaction reagent layer E was disposed on the surface by applying areagent solution, prepared by dissolving 60 mM potassium ferrocyanide,1.0 mass % taurine, and 0.25 mass % maltitol in a 0.1 mass % CMC aqueoussolution, on the surface of the electrode core of the counter electrode(0.55 mg/sensor), and then by drying the solution at 20° C. for 50minutes.

Each of the three kinds of blood samples was introduced into the bloodsample holder of the sensor chip, and a voltage of 2.5 V was appliedacross the working electrode and the counter electrode serving as theanode and the cathode, respectively. A resulting response currentflowing between the working electrode and the counter electrode wasmeasured. The results of measurement of response current are representedby the graph shown in FIG. 25. As shown in the graph, in the sensor chipof Comparative Example 4, the sensitivity difference was unstablethroughout the measurement, even with the application of a relativelyhigh voltage of 2.5 V across the working electrode and the counterelectrode serving as the anode and the cathode, respectively.

Separately, measurements of response current were made as in Examples 1and 2 and Comparative Examples 1 through 4, using sensor chips (1) to(6) for measuring an analyte concentration (described below), and threekinds of blood samples with 25%, 45%, and 65% Hct values, eachcontaining 67 mg/dl of glucose. The results were similar to thoseobtained in the measurements of response current shown in FIGS. 7through 25. Further, a measurement of glucose concentration in the bloodsample using the sensor chips (1) and (2) for measuring an analyteconcentration yielded an accurate result.

The sensor chips (1) through (3) for measuring an analyte concentrationhad the same configurations as the sensor chips of Examples 1 and 2 andComparative Example 1, respectively, except that, in the blood sampleholder, a counter electrode for preliminary measurement having aneffective area of 0.4 mm² was formed to provide a closest distance of1.8 mm between the counter electrode and the counter electrode forpreliminary measurement. The sensor chips (4) through (6) for measuringan analyte concentration had the same configurations as the sensor chipsof Comparative Examples 2 through 4, respectively, except that, in theblood sample holder, a counter electrode for preliminary measurementhaving an effective area of 0.7 mm² was formed to provide a closestdistance of 0.05 mm between the counter electrode and the counterelectrode for preliminary measurement, and that the closest distancebetween the working electrode and the counter electrode was 0.7 mm.

INDUSTRIAL APPLICABILITY

The present invention provides a method for measuring a Hct value of ablood sample, a method for measuring a concentration of an analyte in ablood sample, and a sensor chip and a sensor unit suited for suchmeasurements, that are capable of stably measuring a Hct value of ablood sample with sufficient detection sensitivity even with a small Hctvalue measuring voltage.

The invention claimed is:
 1. A method of determining an amount of ahematocrit in blood using a biosensor, the biosensor comprising: a bloodsample inlet through which a blood sample is introduced, a firstelectrode, a second electrode, an oxidant of a redox substance, and areductant of a redox substance, wherein the reductant of a redoxsubstance is disposed between the blood sample inlet and the firstelectrode, the oxidant of a redox substance is disposed between theblood sample inlet and the second electrode, and all of the oxidant andthe reductant of the redox substance are arranged separately from eachother before delivery of blood sample through the blood sample inlet,with the reductant of the redox substance arranged separately from thefirst electrode and the oxidant of the redox substance arrangedseparately from the second electrode; the method comprising: applying avoltage to the first electrode and the second electrode; detecting acurrent value generated by the application of the voltage; andcalculating an amount of the hematocrit using the current value.
 2. Themethod of claim 1, wherein the voltage is applied for a time of 0.01 to5 seconds.
 3. The method of claim 1, wherein the first and secondelectrode comprise at least one selected from gold, palladium, andcarbon.